The association between adiposity and anti-proliferative response to neoadjuvant endocrine therapy with letrozole in post-menopausal patients with estrogen receptor positive breast cancer

The impact of adiposity on the efficacy of endocrine treatment in patients with estrogen receptor positive breast cancer is poorly investigated. Here, we retrospectively investigated in a cohort of 56 patients whether body mass index and/or mammary adiposity are associated with anti-proliferative response in the neoadjuvant setting. Anti-proliferative response was defined as high Ki67 at baseline (Ki67bl) and low Ki67 at surgery (Ki67srg), using the 14% cut-off. Mammary adipocyte size was assessed on hematoxylin and eosin slides from the surgical samples using digital pathology. A higher proportion of tumors with an anti-proliferative response was observed in patients with obesity (54.5%) as compared to patients with normal weight (9.0%) and patients with overweight (40.0%) (p = 0.031), confirmed by multivariable regression analysis adjusted for baseline Ki67 (OR, obese vs normal weight: 13.76, 95%CI: 1.49–207.63, p = 0.020). Larger adipocyte diameter was identified as predictor of anti-proliferative response (OR per increase in diameter of 5 μm for adipocytes distant from the tumor: 2.24, 95%CI: 1.01–14.32, p = 0.046). This study suggests that anti-proliferative response to neoadjuvant letrozole might be more frequent in patients with increased systemic or mammary adiposity.


INTRODUCTION
The overweight and obese post-menopausal female population is exposed to an increased risk of developing breast cancer (BC) that it is tightly linked to the estrogen receptor (ER)-positive subtype 1 . Increased adiposity, standardly defined by an elevated body mass index (BMI), has prognostic implications, leading to worse outcomes across all BC molecular subtypes 2 . Also, it might affect response to various BC treatments including endocrine treatment (ET), although the underlying mechanisms are mostly still unknown [3][4][5] .
While BMI is a commonly used measure of individual's adiposity, it does not accurately describe the fat mass located in different adipose depots 6 . Recently, the adipocyte size has been used as alternative measure of adiposity, giving information on adiposity in specific body sites such as the mammary adipose tissue 7 . The mammary adipose tissue of women with overweight, obesity and of a non-negligible percentage with normal weight can exhibit a low-grade inflammatory state characterized by adipocyte hypertrophy, macrophage infiltration with the formation of crown-like structures (CLS), and it is associated with a series of changes in the breast adipose tissue microenvironment, including a local increase of aromatase activity 8,9 . During BC initiation and growth, there is a remodeling of the mammary adipose tissue in which cancer cells engage in close contact with adipocytes determining the conversion of adipocytes into the so-called cancer-associated adipocytes (CAAs). CAAs take an active part in the tumor microenvironment, releasing free-fatty acids, inflammatory cytokines, adipokines and other molecules to support the tumor growth 10 .
Most of the patients with ER-positive BC receive ET after surgery 11 . Recently, several neoadjuvant clinical trials have demonstrated that neoadjuvant ET achieves surgical outcomes similar to neoadjuvant chemotherapy with the advantage of less toxicity [12][13][14] . Several trials defined changes in Ki67, measured on pre-treatment and "on-treatment" tumor specimens, as an early biomarker of anti-proliferative response, associated with longterm prognosis [15][16][17] . While inconclusive results have been reported regarding the differential efficacy of ET (aromatase inhibitors-AIs versus tamoxifen), according to patient's BMI in the adjuvant setting 18 , to our knowledge only two studies evaluated the efficacy of AIs in the neoadjuvant setting according to patient's BMI 19,20 . Takada et al. found that the efficacy of exemestane, measured as clinical response, was greater in patients with high BMI compared to patients with low BMI 19 .
In a more recent study, Franzoi et al. showed no significant difference in Ki67 changes and radiological response according to BMI in patients receiving neoadjuvant anastrozole plus abemaciclib 20 . Thus far, no clinical study has investigated the relationship between obesity/mammary adiposity and anti-proliferative response following neoadjuvant letrozole.
In the present study, we retrospectively evaluated whether BMI and the size of the mammary adipocytes are associated with the anti-proliferative response to neoadjuvant aromatase inhibition with letrozole. We further explored whether mammary adiposity was associated with the presence of CLS and tumor-associated macrophages (TAM) and whether mammary adiposity differs according to the main BC histological subtypes (invasive lobular carcinoma-ILC versus invasive carcinoma of no special type -NST, formerly called ductal carcinoma).

Patient characteristics and change in Ki67
In the present study, we considered 56 out of 66 patients that were treated at the Institut Jules Bordet with neoadjuvant letrozole in the context of the Fragrance trial (NCT00199134) (supplementary Fig. 1). Pre-and post-treatment patient and tumor characteristics are summarized in Table 1 according to BMI category. BMI did not show any evidence of association with the standard clinico-pathological characteristics. 13 (23.2%), 20 (35.7%), and 23 (41.1%) out of the 56 patients were normal weight, overweight, and obese, respectively. Distant adipocytes and CAAs were digitally measured in 39 out of 56 (69.6%) and 33 out of 56 (58.9%) patients, respectively. The lower percentage of samples examined for CAAs is justified by the fact that the regions close to the tumor presented globally a minor quality due to fibrosis and damaged adipose tissue. We observed a conversion of ER status from positive to negative from baseline to surgery in 2/ 56 (3.5%) whereas we observed a negative conversion of progesterone receptor (PgR) in 17/46 patients (36.9%). It can be explained by the effect of letrozole that has been reported to significantly reduce the expression of the estrogen-regulated protein PgR 21 . In Supplementary Table 1 we reported the Quick Score for ER and PgR at baseline and at surgery.
Association between BMI and anti-proliferative response No significant association was found between Ki67 bl and BMI (p = 0.263, Fig. 2a) or between Ki67 srg and BMI (p = 0.909, Fig. 2b). We evaluated the association between BMI and anti-proliferative response defined by the "Ki67 bl -Ki67 srg " groups and by Ki67 srg (residual Ki67). Figure 2c shows the distribution of Ki67 bl -Ki67 srg groups according to patients' BMI. We observed a significantly larger proportion of patients with obesity with "high Ki67 bl -low Ki67 srg " tumors (12 out of 22, 54.5%), as compared to patients with overweight (8 out of 20, 40.0%) and patients with normal weight (1 out of 11, 9.0%, p = 0.031). Then, we further explored the relationship between BMI and anti-proliferative response using regression models. We found a significant association between BMI (overweight vs normal weight and obese vs normal weight) and the anti-proliferative response according to the responders vs non responders categorization in the multivariable analysis adjusted for Ki67 bl (odds ratio (OR) obese vs normal weight: 13.76, 95% Confidence Interval (95%CI): 1.48-207.63, p = 0.020, Fig. 2d). A similar association was observed when considering BMI as a continuous variable (OR for 1 kg/m 2 increase: 1.19, 95%CI: 1.00-1.50, p = 0.046, Fig. 2e). Of note, we did not find any evidence of a nonlinear effect for BMI. Similar effects, although not statistically significant, were observed for residual Ki67 and BMI as categorical variable (obese vs normal weight, OR: 3.02, 95%CI: 0.50-20.97, p = 0.223), and residual Ki67 and BMI as continuous variable, at the multivariable level (OR: 1.08, 95%CI: 0.95-1.29, p = 0.230).
Association between adipocyte size, BMI and antiproliferative response First, we compared the median adipocyte area and the median adipocyte diameter of distant adipocytes and CAAs for each patient, and confirmed, in agreement with our previous work 22 , that CAAs were smaller than distant adipocytes (Fig. 3a, b) . Second, we investigated the correlation between BMI and adipocyte size. BMI as categorical variable (normal weight, overweight or obese) was significantly correlated with adipocyte size with regard to area (Kendall's tau = 0.287, p = 0.041, and Kendall's tau = 0.340, p = 0.007, CAAs and distant adipocytes respectively) and diameter (Kendall's tau = 0.304, p = 0.031, Kendall's tau = 0.323, p = 0.011, CAAs and distant adipocytes respectively). The correlation was further confirmed when considering BMI as continuous variable, both with regard to area of CAAs and distant adipocytes (Spearman's rho = 0.355 p = 0.042; Spearman's rho = 0.421 p = 0.007, respectively) and diameter of CAAs and distant adipocytes (Spearman's rho = 0.352 p = 0.044; Spearman's rho = 0.399 p = 0.011, respectively). Third, we explored the association between adipocyte size, BMI and antiproliferative response (Fig. 3c). The "high Ki67 bl -low Ki67 srg " group had larger adipocytes as compared to the "low Ki67 bl -low Ki67 srg " group (p < 0.001, p = 0.012, CAAs and distant adipocytes respectively) and to the "high Ki67 bl -high Ki67 srg " group (p < 0.001, p = 0.007, respectively). Then, we assessed through multivariable regression analysis the relationship between adipocyte size and anti-proliferative response. We found that larger distant adipocyte size (diameter and area) is an independent predictor of antiproliferative response (OR for 5 μm increase in diameter: 2.24, 95% CI: 1.01-14.32, p = 0.046, OR for 100 μm 2 increase in area: 1.17, 95%CI: 1.01-1.74, p = 0.029, Fig. 3g, h). Similar associations were observed for CAAs (Fig. 3i, j). Considering residual Ki67, the direction of association terms were conserved but did not reach statistical significance Association between adipocyte size, BMI, CLS, and TAM We subsequently investigated the presence of CLS in the mammary adipose tissue. CLS were present in the adjacent mammary tissue of 10 out of the 41 patients which could be evaluated (24%; 7 obese, 2 overweight, and 1 normal weight). We found a positive linear correlation between CLS density and adipocyte size ( Supplementary Fig. 2a, b), whereas no significant correlations were found with age (Spearman's rho = 0.132, p = 0.411; all post-menopausal patients) and BMI (Spearman's rho = 0.26, p = 0.100). The presence of CLS (CLS− vs CLS+) was   also associated with a higher distant adipocyte diameter and area ( Supplementary Fig. 2c, d). This association was confirmed by univariable and multivariable linear regression analysis (Supplementary Fig. 2e, f). In the latter, adjusted for continuous BMI, the direction of the estimate is maintained, although it did not reach statistical significance ( Supplementary Fig. 2g, h). Finally, we evaluated the association between TAM categorized in low TAM and high TAM, adipocyte size, and BMI. We showed a positive association, although not significant, between TAM and adipocyte size where increased adipocyte diameter and area were associated with higher presence of TAM (Wilcoxon: p = 0.055, p = 0.067, Supplementary Fig. 3a, b). The same trend was found at the univariable and multivariable level, adjusted for continuous BMI, but the association did not reach statistical significance ( Supplementary Fig. 3c-f). Of note, no association was found between TAM and continuous BMI (IQR: 23.55-34.12, IQR: 29.20-34.65, p = 0.110, low TAM and high TAM respectively), while a positive association was observed between TAM and CLS density (IQR: 0-0, IQR: 0-0.1, p = 0.027, low TAM and high TAM, respectively), as well as between TAM and age (IQR: 60-71, IQR: 68.5-78, p = 0.026 low TAM and high TAM respectively).

Adipocyte size and histological subtypes
We finally explored the association between adipocyte size and BC histology (NST vs ILC; n = 25, n = 10, respectively) and we observed slightly smaller adipocytes in ILC vs NST tumors for both CAAs and distant adipocytes (Fig. 4a-c) Fig. 4d, e for NST and ILC respectively. We further explored this association through univariable and multivariable regression analysis. The direction of the estimates confirmed the aforementioned trend of ILC having smaller adipocytes as compared to NST tumors, but the association did not reach statistical significance (Supplementary Fig. 4).

DISCUSSION
The impact of BMI on ET has been evaluated retrospectively in a few adjuvant clinical trials hypothesizing a lower efficacy of ET in patients with overweight and patients with obesity, but not strongly supported by the evidence [23][24][25] . By contrast, in the neoadjuvant setting, a study consisted of 109 patients, suggested that high BMI might be a positive predictor of AI (exemestane) response based on tumor reduction rate, and another recent study found no difference in Ki67 changes and radiological response according to BMI 19,20 . A large neoadjuvant clinical trial evaluated ET response in post-menopausal ER positive BC patients, using changes in Ki67 before and after 2 weeks of neoadjuvant aromatase inhibition. In the aforementioned trial, four groups of patients were identified, after Ki67 dichotomization, based on Ki67 index at baseline and at 2 weeks. The authors found that a group of patients with the anti-proliferative response to neoadjuvant aromatase inhibition had a better prognosis as compared to the patients whose tumors had still a high Ki67 value at surgery 17  explored the association between anti-proliferative response and BMI/adipocyte size, and BC histology. First, we showed that patients with overweight and patients with obesity had a higher Ki67 suppression following neoadjuvant letrozole as compared to patients with normal weight. We also found that in the obese group there was a higher proportion of patients with high Ki67 bl and low Ki67 srg as compared to patients with overweight and patients with normal weight. In our cohort, higher BMI was associated with anti-proliferative response irrespective of Ki67 at baseline. Considering that BMI is inaccurate in describing the adiposity of specific fat depots, we also assessed mammary adipocyte size as alternative measure of mammary adiposity. In line with our previous work 22 , we showed a correlation between BMI and adipocyte size, although it is moderate. As for BMI, we found that adipocyte size (area and diameter) is a positive predictor of anti-proliferative response, showing that patients with larger distant adipocytes were more likely to have an anti-proliferative response. We also showed that patients with overweight and patients with obesity presented more frequently CLS in the mammary adipose tissue as compared to patients with normal weight, in line with the literature [26][27][28] . Furthermore, there exists a positive correlation between adipocyte size and CLS density confirming that patients with increased adiposity tend to have an inflammatory state, measured as CLS density, in the mammary adipose tissue 7,28 . We also detected a positive association between the presence of TAM and adipocyte size, but not with BMI category. It has been described that ET might contribute to deplete adipocyte progenitors and support the adipocyte hypertrophy only in women with high BMI leading to a higher risk of metabolic diseases. Therefore, increased adipocyte size might be also considered as an early indicator of adverse metabolic effects of endocrine therapies in a specific subgroup of patients 29 .
The association between BMI or adipocyte size on one hand and the anti-proliferative response in BC on the other hand is surprising to some extent. Several studies have consistently reported that postmenopausal patients with a high BMI and ERpositive BC had worse disease outcomes as compared to patients with a lower BMI 2 . We and others have shown that adipocyte size positively correlates with BMI 7,22,30 . This correlation is however not perfect as a subgroup of patients with low BMI also present CLS and larger mammary adipocytes 7 . In our study, we showed that patients with adverse prognostic characteristics such as high BMI, increased adipocyte size, and presence of CLS in the adjacent mammary tissue achieve more frequently an anti-proliferative response to neoadjuvant letrozole, a response which is considered as a favorable prognostic factor 17,31 . Several studies reported a higher aromatase activity in the presence of larger mammary adipocytes and macrophages infiltration 7,26 . We might therefore speculate that the mitogenic effect due to the estrogens, reflected  by the Ki67 index, might be greater in those patients with larger adipocytes and higher aromatase activity as compared to patients with low BMI. Hence, the aromatase inhibition might lead to a greater suppression of the mitogenic effect reflected by the Ki67 reduction in those patients having high BMI and/or larger adipocytes. We acknowledge the complexity of the biological context located at the carrefour between increased adiposity, adipose tissue dysfunction, breast cancer and treatment response.
Our study sheds light on part of the system but the biological meaning through which anti-proliferative response might be positively influenced by BMI and adipocyte size remains to be fully elucidated.
In this study, we also explored the size of mammary adipocytes in the two most prevalent histological subtypes of BC (ILC vs NST). A previous study described a different expression pattern of lipid metabolism proteins between these BC histological subtypes, . c, f Correlation between BMI and distant adipocytes diameter (c) and area (d). Spearman's rho and corresponding P-values are reported on the top-right corner of the plots. The color indicates the Ki67 bl -Ki67 srg groups. Low-low = Ki67 bl and Ki67 srg < 14%. High-low = Ki67 bl ≥ 14% and Ki67 srg < 14%. High-high = Ki67 bl and Ki67 srg ≥ 14%. g-j Multivariable logistic models adjusted for continuous Ki67 bl investigating the association of adipocyte size and the anti-proliferative response: considering distant (g, h) or CAAs i, j and diameter (g, i) or area (h, j). All reported P-values are from Wald tests. BMI body mass index, CAAs cancer-associated adipocytes, CI confidence interval, Ki67 bl Ki67 baseline, Ki67 srg Ki67 surgery, OR odds ratio.
showing a higher expression of certain lipases in ILC 32 .
Hypothesizing that lobular cancer cells may have a stronger interaction with surrounding adipocytes due to looser cell-cell adhesion, we found that CAAs in patients with ILC seems to be smaller as compared to CAAs of patients with NST BC supporting the hypothesis of a higher delipidation in adipocytes from ILC. By contrast, at the histological level, ILC has been described having a fat-avoiding growth 33 . This observation has not been found in our cohort, but we cannot exclude a role of the letrozole in shaping the size of adipocytes and modulating the interaction between cancer cells and CAAs. These observations will need to be further investigated in future studies. Patients and samples were selected based on the present interaction between cancer cells and adipocytes. This means that patients with denser breast parenchyma, in which the tumor has limited contact with the adipose tissue, are not considered in the analysis. Further studies are required to elucidate whether the extent of tumor-adipocyte interaction might influence the biology and anti-proliferative response of the tumor.
There are several limitations in this study that could be addressed in future research. So far, there is no standard definition of anti-proliferative response. We, therefore, used the definitions reported in recent neoadjuvant clinical trials 17,34 . However, one should be careful as various definitions of Ki67 suppression could induce small changes in categorizing patients as responders or non-responders. The number of patients was relatively small, therefore a clinical validation in larger cohorts of patients treated with neoadjuvant AIs is needed. Another aspect was the evaluation of adipocyte size in the pre-treatment biopsies that was only possible for 21 patients (37%) due to small quantity of AT presented on those samples and hence we excluded them from the analysis. Future studies should therefore possibly also consider taking pre-treatment biopsies in the adjacent mammary tissue to allow the evaluation of the adipocytes.
To conclude, our results suggest that letrozole might have a higher anti-proliferative efficacy in patients with increased adiposity. These observations, which need to be confirmed in larger series of patients, further highlight the need of investigating treatment efficacy and BC biology according to the adiposity of the patient.

Patients and slides
The study population was composed by 66 patients treated at the Institut Jules Bordet, Brussels, Belgium. They received four months of neoadjuvant ET with oral letrozole 2.5 mg once daily and underwent surgery between November 2004 and December 2014. Of note, no patients achieved a pathological complete response. The level of expression of ER and PgR was measured by immunohistochemistry using the Quick Score on tumor sample from tru-cut biopsy (pre-treatment) and from surgery specimens (post-treatment) and centrally reviewed as per study protocol (Supplementary Table 1). Patients were eligible for the present sub-study if they met the following criteria: (1) a primary invasive carcinoma of NST or ILC, (2) postmenopausal status at diagnosis, (3) ER positive status of the tumor at diagnosis, (4) available information on BMI recorded at last on the day of oncological surgery. A total of 56 out of 66 patients met the inclusion criteria ( Supplementary Fig. 1). BMI (kg/m 2 ) was categorized according to the World Health Organization (WHO) criteria: normal weight (18.5-24.9 kg/m 2 ), overweight (25-29.9 kg/m 2 ), obese (≥30 kg/m 2 ) 35 . Patients that were underweight (BMI < 18.5 kg/m 2 ) were excluded given their potentially adverse prognosis 36 .
Multiple hematoxylin and eosin (H&E)-stained breast tissue slides, from surgery specimens, were available for adipocyte analysis. After a revision by an expert breast pathologist (D.L.), 112 H&E slides were chosen for subsequent analysis. We excluded the slides with damaged adipose tissue or low quality of the staining. H&E-stained slides were scanned using a Nanozoomer digital scanner (c10730-12, Hamamatsu) with a 40x objective (0.228 mm/pixel). Mammary adipocytes (distant from the tumor and CAAs) were selected and annotated in digital slides as follows: adipocytes distant from the tumor were defined as those being at least 2 mm away from cancer cells as well as 2 mm away from fibrosis area and epithelial structures. CAAs were defined as the three first lines of adipocytes at the invasive front of the tumor and as maximum 2 mm within the tumor starting from the invasive front 22 . A minimum of 500 individual distant adipocytes and 500 individual CAAs per patient were measured to determine median adipocyte area (μm²) and median adipocyte diameter (μm) using HALO ® software, version 2.3 (vacuole module, version 2.2 -Indica Labs, Corrales, NM, USA). Measurements with a diameter under 30 μm were not considered in our analysis in order to exclude artefacts. Immunohistochemistry for CD68 was performed on sections from formalin-fixed paraffin-embedded tumor blocks, using the monoclonal mouse anti-human CD68 antibody (clone KP1), ready to use (prediluted). An expert breast pathology (D.L.) counted and recorded the number of CLS in the adjacent mammary tissue. The CLS density (CLS/cm 2 ) was calculated as the number of CLS per square centimeter of adipose tissue. On average 5 blocks per patient were stained for CD68. CD68-positive cells were further counted and recorded, as percentage of TAM in each scanned tumor slide. TAM were categorized into low or high category according to 10% cut-off.
The project has been approved by the ethics committee of the Institut Jules Bordet (CE2844, 3 May 2018) and all patients provided written informed consent.

Ki67 assessment
Centralized Ki67 assessment was performed. Ki67 delta suppression was defined as the difference between Ki67 at surgery and at baseline (Ki67 srg -Ki67 bl ). The primary endpoint was to evaluate the anti-proliferative response. Comparing Ki67 levels at baseline with those at surgery after Ki67 dichotomization using a 14% cut-off, four groups of patients were identified: "low Ki67 bl -low Ki67 srg " (Ki67 bl and Ki67 srg < 14%); "high Ki67 bl -low Ki67 srg " (Ki67 bl ≥ 14%, Ki67 srg < 14%); "high Ki67 bl -high Ki67 srg " (Ki67 bl and Ki67 srg ≥ 14%); and "low Ki67 bl -high Ki67 srg " (Ki67 bl < 14%, Ki67 srg ≥ 14%). Responders were the patients in the "high Ki67 bl -low Ki67 srg " group, "high Ki67 bl -high Ki67 srg " were considered as nonresponders while "low Ki67 bl -low Ki67 srg " were excluded from the analysis. No patients were classified into the "low Ki67 bl -high Ki67 srg " group. An alternative measure of response was used, and it was defined by the Ki67 index at surgery, also called residual Ki67. Responders and non-responders were identified as the patients with a residual Ki67 value of <14% or ≥ 14%, respectively. These definitions were chosen for consistency with previous neoadjuvant clinical trials. Three patients were excluded from the analysis because of missing Ki67 values.
The PEPI consisting of pathological tumor size, pathological node status, Ki67 index, and ER status of residual tumors after neoadjuvant ET, was assessed according to the algorithm of Ellis 16 . PEPI score was categorized into three risk groups as follows: low (0 points), moderate (1-3 points), and high (≥4 points). 1 out of the 56 patients (1.9%) had a PEPI score = 0, 29 out of 56 patients (53.7%) had a PEPI score between 1 and 3, and 24 out of 56 patients (44.4%) had PEPI score ≥ 4.

Statistical Analysis
We evaluated the association between baseline clinico-pathological characteristics and BMI using Fisher exact test and Kruskal-Wallis test when comparing categorical and continuous variables against the three BMI categories. Associations between continuous and (ordinal) categorical variables were assessed using Wilcoxon and Mann-Kendall tests. Correlation between continuous BMI and continuous adipocyte measurements was assessed using the Spearman rank test. Univariable and multivariable Firth logistic regression models with anti-proliferative response or residual Ki67 as the response variables, BMI (continuous and categorical), adipocyte size (diameter and area) as the predicting variables of interest, and Ki67 bl as covariate in the anti-proliferative response, were used to assess the associations between BMI, adipocyte size, and anti-proliferative response or residual Ki67. In the continuous setting, non-linear effects were explored during the model-building phase resorting to regression penalized cubic splines. In a similar manner, we also performed multivariable linear regression analysis, adjusted for BMI, to evaluate the association of adipocyte size with CLS presence (CLS+ vs CLS−), TAM category (high vs low), and histological BC subtype (ILC vs NST). All odds ratios (OR) were computed for 1 kg/m 2 increase in BMI but for 5 μm and 100 μm 2 for adipocyte's diameter and area respectively. Patients with missing values were excluded from the analysis. The Wilcoxon rank-sum test was performed to evaluate the association between adipocyte size and histology. The distribution of BMI in patients with adipocyte size data included in the multivariable analysis (n = 41) was not significantly different from the whole patient population (n = 56), nor from that in the excluded patients. P-values were 2-sided and considered as statistically significant at the conventional level of .05. Consistently, 95% confidence intervals (CIs) were appropriately computed with the functions provided in the logistf, and rms packages. Statistical analyses were performed using R version 4.0.2 37 .

Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.

DATA AVAILABILITY
The data that support the findings of this study are available upon request to the corresponding author after signature of a Data Access Agreement. The data are not publicly available due to the personal nature of the containing information.